Open Access
Research article
Reinterpreting a community outbreak of Salmonella enterica
serotype Enteritidis in the light of molecular typing
Cristina Romani
1, PierLuigi Nicoletti
2, Maria Ida Buonomini
2,
Antonino Nastasi
1and Caterina Mammina*
3Address: 1Department of Public Health, University, Via G.B. Morgagni 48, 50134 Florence, Italy, 2Laboratory of Microbiology and Virology, Azienda Ospedaliero-Universitaria Careggi, Via G.B. Morgagni 85, 50134 Florence, Italy and 3Centro per gli Enterobatteri Patogeni dell'Italia Meridionale (CEPIM), Department of Hygiene and Microbiology, University, Via del Vespro 133, 90127 Palermo, Italy
Email: Cristina Romani - cristina.romani@unifi.it; PierLuigi Nicoletti - nicolettip@ao-careggi.toscana.it; Maria Ida Buonomini - labmicro20@ao-careggi.toscana.it; Antonino Nastasi - antonino.nastasi@unifi.it; Caterina Mammina* - diptigmi@unipa.it
* Corresponding author
Abstract
Background: In November 2005, a large outbreak due to Salmonella enterica serotype Enteritidis
(S. Enteritidis) was observed within children who had eaten their meals at 53 school cafeterias in Florence and the surrounding area. A total of 154 isolates of S. Enteritidis were recovered from human cases between November 2005 and January 2006. All strains were assigned phage type 8 (PT8) and a common XbaI pulsotype.
This paper reports the findings of a molecular epidemiological investigation performed on 124 strains of S. Enteritidis isolated in the years 2005 and 2006 in Florence and the surrounding area, including the epidemic isolates.
Methods: One hundred twenty-four human isolates of S. Enteritidis identified in the period
January 2005 – December 2006 were submitted to molecular typing by single enzyme – amplified fragment length polymorphism (SE-AFLP).
Results: Molecular subtyping by SE-AFLP yielded five different profiles. In the pre-epidemic phase,
type A included 78.4% of isolates, whereas only three (8.1%) belonged to type C. All isolates, but one, of the epidemic phase were indistinguishable and attributed to type C. In the post-epidemic period, a polymorphic pattern of SE-AFLP types was again recognized but type C accounted for 73.3% of the isolates during the first six months of 2006, whereas during the remaining six months type A regained the first place, including 52.0% of the isolates.
Conclusion: The epidemic event was attributed to the emergence and clonal expansion of a strain
of S. Enteritidis PT8-SE-AFLP type C. Circulation of the epidemic clone was much more extensive than the surveillance and traditional laboratory data demonstrated.
Background
Salmonellosis resulting from the ingestion of
contami-nated food of animal and non animal origin, is a signifi-cant public health problem worldwide [1,2]. Foodborne
Published: 7 September 2007
BMC Public Health 2007, 7:237 doi:10.1186/1471-2458-7-237
Received: 8 May 2007 Accepted: 7 September 2007 This article is available from: http://www.biomedcentral.com/1471-2458/7/237
© 2007 Romani et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
disease outbreaks due to Salmonella serotypes are very common in most countries, where this organism has in many cases took advantage from the dramatic changes occurring in the food production chain through the last decades [1,3-6].
Salmonella enterica subsp. enterica serotypeTyphimurium
(S. Typhimurium) and S. Enteritidis are by far the most prevalent serotypes in humans [2]. S. Enteritidis is the most common Salmonella serotype globally but especially in Europe, where it accounts for approximately 80–85% of Salmonella cases [2]. In particular, Enteritidis has emerged in Italy since the eighties in association with egg-borne outbreaks, consistently with similar findings of a worldwide increase in S. Enteritidis infections. Moreover, in many European countries, including Italy, after a decline between the late 1990s and 2002 due to imple-mentation of more stringent European legislation about breeding flocks and improved sanitation and farming practices for egg-laying hens and broilers, the incidence of human infections has again increased starting during the last quarter of 2002 in association with a shift in the prev-alent phage types [7,8]. Some countries, like England and Wales and Luxembourg have recently reported a substan-tial reduction of infection cases due to this serotype [9,10].
Since November 2005, the Azienda Sanitaria Locale of Florence observed a dramatic increase of isolation of S. Enteritidis from paediatric cases occurring in Florence and the surrounding area. Public Health Services of Florence, with the technical support of the microbiology laboratory of the "Azienda Universitario-Ospedaliera Careggi" and the Istituto Zooprofilattico Sperimentale of Lazio and Tuscany, started an epidemiological investigation. Chil-dren involved in the epidemic proved to be pupils of 53 different schools, who had eaten their meals at multiple school cafeterias contracted out with the same external caterer [11].
The epidemic curve showed a prominent wave consistent with a point common source and a prolonged downslope of the curve including December 2005 and January 2006, that was interpreted as the result of secondary transmis-sion [11].
A total of 154 isolates of S. Enteritidis were recovered from human cases between November 2005 and January 2006. Two food handlers tested also positive for S. Enteritidis. Attempts to identify food vehicle and trace route of trans-mission were unsuccessful. All strains were assigned phage type 8 (PT8) and were included into a common
XbaI pulsotype by pulsed field gel electrophoresis (PFGE)
[11].
We report the findings of a molecular epidemiological investigation performed on 124 strains of S. Enteritidis isolated in the years 2005 and 2006 in Florence and the surrounding area, including the epidemic isolates. Char-acterization of the strains has been performed by single enzyme – amplified length polymorphism (SE-AFLP), a molecular subtyping method previously applied on a large selection of sporadic and outbreak isolates of the serotype Enteritidis [12]. The study has been carried out in order to evaluate molecular epidemiological features of the epidemic clone in comparison with the strains recov-ered from apparently sporadic human cases of infection before and after the epidemic peak.
Methods
Bacterial strains
One hundred twenty-four isolates of S. Enteritidis identi-fied from human cases of infection in the period January 2005 – December 2006 at the laboratory of Microbiology of the "Azienda Universitario-Ospedaliera Careggi" have been submitted to molecular typing.
All isolates used in this study had been previously identi-fied by the API20E system [Biomerieux, Marcy l'Etoile, France], serotyped by commercially available antisera [Statens Serum Institut, Copenhagen, Denmark] and stored at -70°C.
The strains were classified into three groups: pre-epidemic (January – first week of November 2005), epidemic (sec-ond week of November 2005 – January 2006) and post-epidemic (February – December 2006). All available iso-lates recovered in the pre- and post-epidemic phases, respectively, 37 and 55 isolates, were included in the study. Moreover, 30 isolates were selected by systematic sampling among the 154 isolates from the cases occurring throughout the entire epidemic interval of time and cul-tured at the "Azienda Universitario-Ospedaliera Careggi". The isolates were stratified by week of isolation and, then, every fifth isolate of the week was included in the study. Both S. Enteritidis isolates from the two food handlers were added.
SE-AFLP
After extraction, DNA was submitted to a simultaneous reaction of digestion with the restriction endonuclease
HindIII and ligation at 37°C for 3–4 h. Adapters H1,
5'-ACGGTATGCCACAG-3' and H2, 5'-AGCTCTGTCGCAT-ACCGTGAG-3' were used. Digested-ligated DNA was heated to 80°C for 10 min to inactivate the ligase, and subsequent PCR was carried out in a 50 µl amplification mixture containing 200 mM each deoxynucleoside tri-phosphate (dNTP), 2.5 mM MgCl2, 150 ng of each primer and 1 U of Taq polymerase.
A mix of four primers was used for polymerase chain reac-tion (PCR). The primers had the sequence 5'-GGTAT-GCGACAGAGCTTX-3', where X was either A, T, G or C, respectively. PCR amplification consisted of 1 cycle at 94°C for 4 min, followed by 33 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 2.5 min. PCR products were detected on a 1.5% agarose gel run alongside a 100 bp lad-der at 100 V for 3 h, followed by staining in ethidium bro-mide. Photos of the gels were taken under UV light and the gel images were captured in image files. SE-AFLP pro-files were analyzed, identified and compared by visual inspection. A difference in at least one band in the range of 600 – 200 bp was adopted as the criterion for type dif-ferentiation. Profiles were then classified and labelled using alphabetical letters according to a previously used identification scheme [12].
Results
Molecular subtyping by SE-AFLP of the 124 S. Enteritidis isolates under study yielded five different profiles (Fig. 1).
Types A and C were the most prevalent patterns, includ-ing, respectively, 37.9% and 51.6% of the strains. SE-AFLP profiles A and C had been previously recognized as the most frequent types SE-AFLP among a large selection of sporadic and epidemic isolates from different regions of Italy [12]. Three new types were also identified and named, respectively, P, T and Z. Only 13 isolates were attributed to these low-prevalence SE-AFLP types.
Analysis of the relative prevalence of the five types in the three periods – pre-epidemic, epidemic and post-epi-demic – revealed some relevant differences.
In the pre-epidemic phase, type A included 78.4% of iso-lates under examination, whereas only three (8.1%) belonged to type C and four (10.8%) and one (1.7%), respectively, to types P and T.
SE-AFLP types detected during the period January 2005 – December 2006 Figure 1
SE-AFLP types detected during the period January 2005 – December 2006.
MW type P type T type C type A type Z MW
700
600
500
400
200
300
bp
All isolates, but one, of the epidemic phase were indistin-guishable and attributed to type C. The single isolate of type A was recovered from a 91 year-aged woman. In the post-epidemic period, a polymorphic pattern of SE-AFLP types was again recognized: type C accounted for 30 (54.5%) isolates, whereas 17 (30.9%) were attributed to type A, four (7.2%) to type P and one (1.8%) to type T. Within type C isolates, eight isolates were retrospectively identified as belonging to a household epidemic cluster occurred in July 2006. A new profile, named Z, was iden-tified in isolates from three members of the same family. Of interest, type C was predominant during the first six months of 2006, accounting for 73.3% of the isolates, whereas during the remaining six months type A regained the first place, including 52.0% of the isolates. However, in this last period, 32.0% of isolates still belonged to type C. Figure 2 illustrates the temporal trend of detection of the SE-AFLP types throughout the interval of time under study.
Discussion
Since its emergence, S. Enteritidis is challenging pheno-typic and molecular typing methods, because of the great
homogeneity in the genetic make-up of most prevalent strains [12-15]. Time-space defining outbreak events, trac-ing transmission routes, buildtrac-ing microbiologically con-firmed causal relationship between human cases and food vehicles have proved to be a very frustrating exercise when dealing with such a serotype [12-15]. Indeed, discrimina-tory power of the typing techniques has often be unable to reliably differentiate epidemiologically unrelated strains and, eventually, the prominent role of eggs and poultry products as the generally incriminated food items has to some extent withdrawn the interest from a finest strain characterization.
SE-AFLP, a PCR based DNA fingerprinting technique that showed favourable results when applied on a large number of strains of S. Enteritidis in terms of discrimina-tory power, userfriendliness and cost in comparison with phage typing and PFGE, has been applied in this study [12]. Moreover, in the previous study, despite the subop-timal discriminatory power of the technique when applied as a single subtyping approach, a very specific association between SE-AFLP type C and PT2/8 was clearly evident [12].
Distribution of SE-AFLP profiles during the pre-epidemic, epidemic and post-epidemic periods Figure 2
Distribution of SE-AFLP profiles during the pre-epidemic, epidemic and post-epidemic periods.
0
10
20
30
40
50
60
70
80
90
100
% of i sol at estype C
type A
type P
type T
type Z
01 jan - 07 nov 2005 08 nov – 31 jan 2006 01 feb - 31jul 2006 01 aug – 31 dec 2006Some considerations can be drawn from the results of this investigation:
a) the epidemic event was attributable to the emergence and clonal expansion of a strain, that previously was infre-quently playing the role of causative agent of enteritis in the geographic area under study. Type C in the pre-epi-demic phase had a low prevalence.
b) phage typing, that had been previously applied to all strains isolated during the epidemic peak, included them into PT8. It seems reasonable to conclude that the associ-ation PT8-SE-AFLP C characterizes the epidemic clone. c) the post-epidemic phase appears to be again character-ized by a polymorphic pattern, where type C persisted in a prominent position during the first half-year, whereas in the following months type A went back to become again the most frequently identified profile.
d) though in an apparently declining stage, type C was responsible for at least one outbreak in the last months of 2006.
Emergence of type C after a period of low prevalence in the geographic area of interest coincided with an explosive outbreak traced to school cafeterias and, ultimately, to a single caterer. However, beyond the evidence arising from the conventional epidemiological investigations, circula-tion of the epidemic clone has likely been much more extensive than the surveillance and traditional laboratory data suggested. Indeed, molecular typing support the hypothesis that the epidemic phase did not stop at the end of January 2006. On the contrary, S. Enteritidis type C seems to have been protagonist of a hyper-endemic/epi-demic exploit until at least the first months of 2006. The slow downslope of the epidemic curve should be reinter-preted based upon the molecular epidemiological evi-dence: indeed, the previously defined secondary cases appear to arise more likely from a common source that continued over time through the next months.
Moreover, these data strongly support a recent sustained circulation in the geographic area of interest of PT8, his-torically characterized in Italy as a consistently low preva-lence phage type [12].
Conclusion
Molecular subtyping can significantly contribute to a more reliable interpretation of epidemiological events. Final confirmation of the existence of an outbreak should rely on collaborative interpretation of molecular and con-ventional epidemiologic data together. Moreover, deter-mining dynamics of disease transmission in widespread areas, distinguishing epidemic from endemic occurrence
of transmissible disease and, ultimately, identifying and assessing new and more effective control options could greatly benefit from this synergic approach.
Competing interests
The author(s) declare that they have no competing inter-ests.
Authors' contributions
AN, PN and MIB conceived the study. PN and MIB carried out isolation and identification of bacterial isolates. CR performed SE-AFLP typing and analysis of genetic similar-ity. CM participated in study design, analysis of results and collaborated with AN to draw the conclusions and draft the manuscript. All authors read and approved the final manuscript.
Acknowledgements
CR is the recipient of a PhD grant from the PhD School "Pediatria clinica e preventiva", University of Florence, Italy.
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